p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver

Background: Clinically, the persistence of HBV cccDNA is the major obstacle in anti-HBV therapy. However, the underlying mechanism of HBV cccDNA is poorly understood. The transcriptional factor STAT3 is able to activate HBV replication in liver. Approach & Results: RNA-Seq analysis demonstrated that cucurbitacin I targeting STAT3 was associated with virus replication in liver. HBV-infected human liver chimeric mouse model and HBV hydrodynamic injection mouse model were established. Then, we validated that cucurbitacin I effectively limited the stability of HBV cccDNA and HBV replication in cells, in which cucurbitacin I enhanced the sensitivity of pegylated interferon α (PEG-IFN α) against HBV via combination in vitro and in vivo. Mechanistically, we identified that cucurbitacin I increased the levels of APOBEC3B to control HBV cccDNA by inhibiting p-STAT3 in cells, resulting in the inhibition of HBV replication. Moreover, RNA-Seq data showed that E3 ubiquitin ligase DTX4 might be involved in the events. Then, we observed that HBV particles could upregulate DTX4 by increasing the levels of p-STAT3 in vitro and in vivo. The p-STAT3-elevated DTX4/male-specific lethal 2 (MSL2) independently and synergistically enhanced the stability of HBV cccDNA by facilitating the ubiquitination degradation of APOBEC3B in cells, leading to the HBV replication. Conclusions: p-STAT3-elevated DTX4 confers the stability of HBV cccDNA and HBV replication by facilitating the ubiquitination degradation of APOBEC3B. Cucurbitacin Ⅰ effectively enhances the sensitivity of PEG-IFN α in anti-HBV therapy by inhibiting the p-STAT3/DTX4/MSL2/APOBEC3B signalling. Our finding provides new insights into the mechanism of HBV cccDNA. The p-STAT3 and DTX4/MSL2 might serve as the therapeutical targets of HBV cccDNA.

The HBV particles used in this study were mainly derived from HepAD38 cells, and prepared and quantified by a previously described method [4,5].HBV infection was performed as previously described [2,3,5].

RNA extraction and quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from cells (or liver tissues from human liver chimeric mice) using TRIzol reagent (Solarbio, Beijing, China).First-strand cDNA was synthesized using Hifair Ⅲ 1st Strand cDNA Synthesis SuperMix (Yeasen Biotech, Shanghai, China).Quantitative real-time PCR was performed in a StepOnePlus real-time PCR system (Bio-Rad) using SYBR Green qPCR Master Mix (Yeasen Biotech, Shanghai, China).Relative fold differences in transcription were calculated by the 2 -ΔΔCt method.GAPDH was used as the internal control for normalization.The primers used are listed in Supporting Information Table 4.

Dual-luciferase reporter assay
Cells were transferred into 24-well plates at 3× 10 4 cells/well.After 12 h, the cells in each well were transiently co-transfected with 0.2 μg of the pRL-TK plasmid (Promega, Madison, WI, USA) containing the Renilla luciferase gene used for internal normalization and the pGL3-DTX4 promoter, pGL3-MSL2 promoter, pGL3-DTX4 promoter mut or pGL3-MSL2 promoter mut plasmid (lost the ability to interact with STAT3, termed mutant luciferase reporter) (Supporting Information Table 5).Luciferase activities were measured as previously described [6].All experiments were performed at least three times.

HBV cccDNA extraction and quantification
The procedure for the extraction of HBV cccDNA from HepAD38 (tet-off) has been reported previously [4,5].Briefly, aliquots of each DNA sample extracted from cell pellets were treated for 1 hour at 37 °C with 10 U of Plasmid-Safe ATP-Dependent DNase (Epicentre, Madison, WI).qPCR analysis was carried out in a Mastercycler ep realplex instrument (Eppendorf, Germany) to quantify the cccDNA.The primers used in this experiment are listed in Supporting Information Table 4.

Quantification of HBV antigens and serum HBV DNA
Hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in culture supernatants were assayed with commercial enzyme-linked immunosorbent assay (ELISA) kits (Kehua Bioengineering, Shanghai, China).HBV DNA in the supernatants of HepAD38 cells and HBV-infected HepG2-NTCP cells was quantified by using a diagnostic kit with a lower limit of detection of 400 copies/mL (Sansure Biotech, Changsha, China).

Immunofluorescence analysis
Cells in 6-well plates were washed three times with precooled PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 10 min at room temperature.After incubation for 1 h with 5% BSA to block nonspecific binding, primary antibodies were added for incubation overnight at 4 °C.The bound antibodies were detected by incubation with secondary antibodies.Images were acquired using fluorescence microscopy.The antibodies used for immunofluorescence analysis are listed in Supporting Information Table 6.

Co-immunoprecipitation (Co-IP)
Whole-cell extracts were prepared using IP lysis buffer (Beyotime) supplemented with a complete protease inhibitor cocktail (Solarbio) according to the manufacturer's instructions.
Cell lysates were then incubated with the IP antibody overnight at 4 ℃ on a rotating wheel before they were incubated with Protein A/G magnetic beads (Millipore) for 4 h at 4 ℃ on a rotating wheel.The PPIs were analysed by immunoblotting.

Immunohistochemical (IHC) staining
Mice were sacrificed, and their liver tissues were fixed and embedded in paraffin.First, the sections were dewaxed.Then, antigen retrieval was performed at 95 °C with citrate buffer (pH 6.0) for 15 min.The slides were treated with 3% H2O2 for 10 min and blocked with goat serum for 1 h.Then, the slides were incubated with a monoclonal antibody at 4 °C overnight.
After washing three times with 0.01 M PBS, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (OriGene, Beijing, China) for 30 min at 37 °C.Immunohistochemistry staining was performed using the chromogen 3,3′-diaminobenzidine (DAB), and counterstaining was performed with Mayer's haematoxylin (ZSGB-BIO, China).The sections were then dehydrated and covered with a coverslip.The antibodies used for IHC staining are listed in Supporting Information Table 6.

Chromatin immunoprecipitation (ChIP)
ChIP was performed by using a SimpleChIP® Plus Sonication CHIP Kit 4C and RT reagents according to the manufacturer's instructions (Cell Signaling Technology, MA, USA) [7].
DNA synthesis was analyzed by quantitative PCR.Precipitated DNA was analyzed by quantitative PCR, and the primers used are listed in Supporting Information Table 4.

Western blot analysis
Total protein was extracted by lysis of hepatoma cells or tissues with RIPA buffer.Protein concentrations were measured using the Bradford assay, and 20-50 μg of protein extracts were subjected to SDS-PAGE.Then, proteins were transferred to a nitrocellulose membrane, blocked with 5% nonfat milk and incubated with primary antibodies overnight at 4 °C.After incubation with an anti-mouse (1:10,000) or anti-rabbit (1:10,000) secondary antibody for 1 h at 37℃, immunocomplexes were visualized with Super ECL Detection Reagent (Yeasen Biotech, Shanghai, China).The primary antibodies used for Western blot analysis are listed in Supporting Information Table 6.

RNA-Seq analysis
The cells were collected in TRIzol reagent, and RNA-Seq analysis was performed at Shanghai Majorbio Bio-pharm Technology Co. Ltd.The data were analyzed on the free online platform Majorbio Cloud Platform (https://www.majorbio.com/).Genes with an FDR-adjusted P < 0.05 were considered significantly differentially expressed.

Bioinformatics analysis
The public web servers JASPAR (https://jaspar.elixir.no/) was used to conduct bioinformatics analysis, JASPAR was applied to the binding sites of transcription factor on the STAT3, DTX4 or MSL2 promoter.Analysis was performed 7 days post-infection.qPCR analysis was performed to determine whether the effect of STAT3 on the level of HBV cccDNA through MSL2 in the cells (G).
ChIP-qPCR analysis was performed to determine whether the effect of STAT3 on binding of APOBEC3B to HBV cccDNA micro-chromosome through MSL2 in the cells (H).The mean ± SD of at least three experiments is shown.Statistically significant differences are indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001.Abbreviation: A3B, APOBEC3B.

FIGURE
FIGURE S2 Cucurbitacin I attenuates the HBV replication and enhances the sensitivity of

FIGURE S3
FIGURE S3The effect of Cucurbitacin I on APOBEC3A and APOBEC3B.(A) The mRNA

FIGURE S7
FIGURE S7 STAT3 enhances the stability of HBV cccDNA and HBV replication through

FIGURE
FIGURE S8 Cucurbitacin I limits the expression of APOBEC3B by inhibiting

Table 2 .
Clinical characteristics of 60 pairs of clinical liver cancer tissues.

Table 3 .
The information of mice with humanized liver.

Table 4 .
List of Oligonucleotide Sequences used in this article.

Table 5 .
List of plasmids used in this article.

Table 6 .
List of antibodies used in this article.